LOCUS       sequence-237166-       13424 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SP-dCas9-KRAB-MeCP2 with doxycycline-inducible expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13424)
  AUTHORS   Tan X, Worley J, Turunen M, Wong K, Fernandez EC, Paull E, Jones S, 
            Wang J, Noh H, Salvatori B, Chavez A, Califano A
  TITLE     Interrogation of genome-wide, experimentally dissected gene 
            regulatory networks reveals mechanisms underlying dynamic cellular 
            state control
  JOURNAL   bioRxiv 2021.06.28.449297
REFERENCE   2  (bases 1 to 13424)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2021.06.28.449297"
COMMENT     Sequence Label: PB-TRE-dCas9-KRAB-MeCP2
FEATURES             Location/Qualifiers
     source          1..13424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     80..104
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      198..207
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(204..230,231..233,234..2717,2718..2723,2724..2789,
                     2790..2792,2793..4307)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4311..4337
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4317..4343
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             4356..4376
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4383..4403
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4443..4628
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             4662..4682
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4692..5549
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     primer_bind     complement(5604..5623)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5648..5696
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        5927..6138
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             6151..6419
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             6453..7196
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             7197..7250
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             7272..8291
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             7272..8291
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(8348..8368)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    8402..8523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8439..8458)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8493..8512
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9286..9320
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9811..9827)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9811..9827)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9824..9846)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9835..9851
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(9859..9865,9866..9883,9884..9889))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9904..9925
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10042..10059)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10213..10801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10293..10312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10972..11763,11764..11832))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     11595..11614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11833..11937)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11963..12418)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12100..12121)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12312..12331
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12545..12567
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12559..12576
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12560..12576
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12706..12768
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        join(13217..13424,1..171)
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    13225..13243
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13261..13279
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13297..13315
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13333..13351
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13369..13387
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13405..13423
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 cgtatatcca gtttactccc tatcagtgat agagaacgta taagctttag gcgtgtacgg
       61 tgggcgccta taaaagcaga gctcgtttag tgaaccgtca gatcgcctgg agcaattcca
      121 caacactttt gtcttatacc aactttccgt accacttcct accctcgtaa aggtctagag
      181 ctagcgaatt cgaatttgcc accatggaca agaagtactc cattgggctc gctatcggca
      241 caaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca
      301 aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc gccctcctgt
      361 tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata
      421 cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg
      481 tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc
      541 acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc
      601 caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct gacttgcggt
      661 tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc atcgaggggg
      721 acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt cagacttaca
      781 atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa gcaatcctga
      841 gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga
      901 agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta
      961 aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac acctacgatg
     1021 atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg
     1081 caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca
     1141 ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga
     1201 ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg
     1261 atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt
     1321 acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc
     1381 ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc
     1441 agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt
     1501 tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag
     1561 gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca
     1621 tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg
     1681 aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc
     1741 tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag
     1801 ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct
     1861 tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg
     1921 aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca tccctgggaa
     1981 cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat gaggagaacg
     2041 aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg gagatgattg
     2101 aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa cagctcaaga
     2161 ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg atccgagaca
     2221 agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc aaccggaact
     2281 tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag aaagcacaag
     2341 tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt agcccagcta
     2401 tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa gtaatgggaa
     2461 ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact acccagaagg
     2521 gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa gaactggggt
     2581 cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag aagctctacc
     2641 tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac atcaatcggc
     2701 tctccgacta cgacgtggct gctatcgtgc cccagtcttt tctcaaagat gattctattg
     2761 ataataaagt gttgacaaga tccgataaag ctagagggaa gagtgataac gtcccctcag
     2821 aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc aaactgatca
     2881 cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct gagttggata
     2941 aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag cacgtggccc
     3001 aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg attcgagagg
     3061 tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac tttcagtttt
     3121 ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg aatgcagtgg
     3181 taggcactgc acttatcaaa aaatatccca agcttgaatc tgaatttgtt tacggagact
     3241 ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata ggcaaggcca
     3301 ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag attacactgg
     3361 ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg
     3421 tgtgggacaa gggtagggat ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga
     3481 acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt atcctcccga
     3541 aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag aaatacggcg
     3601 gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg gagaaaggga
     3661 agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa
     3721 gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg atataaagag gtcaaaaaag
     3781 acctcatcat taagcttccc aagtactctc tctttgagct tgaaaacggc cggaaacgaa
     3841 tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc tctaaatacg
     3901 ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc gaagataatg
     3961 agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc atcgagcaaa
     4021 taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag gtgctttctg
     4081 cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt atccacttgt
     4141 ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc accatagaca
     4201 gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat cagtcaatta
     4261 cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacgcc tatccctatg
     4321 acgtgcccga ttatgccagc ctgggcagcg gctcccccaa gaaaaaacgc aaggtggaag
     4381 atcctaagaa aaagcggaaa gtggacggca ttggtagtgg gagcaacggc agcagcggat
     4441 cccggacact ggtgaccttc aaggatgtat ttgtggactt caccagggag gagtggaagc
     4501 tgctggacac tgctcagcag atcgtgtaca gaaatgtgat gctggagaac tataagaacc
     4561 tggtttcctt gggttatcag cttactaagc cagatgtgat cctccggttg gagaagggag
     4621 aagagccctc gggaggtggt tcgggtggct ctggatcaag cccaaagaag aaacggaagg
     4681 tggaagcctc agtgcaggtg aaaagggtgc tggaaaaatc ccccggcaaa ctcctcgtga
     4741 agatgccctt ccaggcttcc cctggcggaa aaggtgaagg gggtggcgca accacatctg
     4801 cccaggtcat ggtcatcaag cgacctggaa ggaaaagaaa ggccgaggct gaccctcagg
     4861 ccattccaaa gaaacgggga cgcaagccag ggtccgtggt cgcagctgca gcagctgagg
     4921 ctaagaaaaa ggcagtgaag gaaagctcca tccgcagtgt gcaggagact gtcctgccca
     4981 tcaagaagag gaagactagg gagaccgtgt ccatcgaggt caaagaagtg gtcaagcccc
     5041 tgctcgtgtc caccctgggc gaaaaatctg gaaaggggct caaaacatgc aagtcacctg
     5101 gacggaaaag caaggagtct agtccaaagg ggcgctcaag ctccgcttct agtcccccta
     5161 aaaaggaaca ccatcaccat caccatcacg ccgagtctcc taaggctcct atgccactgc
     5221 tcccaccacc tccaccacct gagccacagt caagcgaaga ccccatcagc ccacccgagc
     5281 ctcaggatct gtcctctagt atttgcaaag aggaaaagat gcccagagca ggcagcctgg
     5341 agagtgatgg ctgtccaaaa gaacccgcca agacccagcc tatggtggca gccgctgcaa
     5401 ctaccaccac aaccacaact accacagtgg ccgaaaaata caagcatcgc ggcgagggcg
     5461 aacgaaagga cattgtgtca agctccatgc ccagacctaa ccgggaggaa ccagtcgata
     5521 gtaggacacc cgtgactgag agagtctcat gaaagggttc gatccctacc ggttagtaat
     5581 gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     5641 gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt ttgttcataa
     5701 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
     5761 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
     5821 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcagatctgc gaaatcggat
     5881 ccagctaaat ctaagtggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca
     5941 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga
     6001 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     6061 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     6121 tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc
     6181 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt
     6241 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc
     6301 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg
     6361 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatct
     6421 aagctgtgac cggcgcctac tctagagccg ccatgtctag actggacaag agcaaagtca
     6481 taaactctgc tctggaatta ctcaatggag tcggtatcga aggcctgacg acaaggaaac
     6541 tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg
     6601 ccctgctcga tgccctgcca atcgagatgc tggacaggca tcatacccac tcctgccccc
     6661 tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcatac cgctgtgctc
     6721 tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt
     6781 acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg
     6841 cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg
     6901 agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc
     6961 tgaaacaagc aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc
     7021 tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccgaccg
     7081 acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc
     7141 ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc cccggggagg
     7201 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct gaattcatgg
     7261 atagatccgg aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     7321 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     7381 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     7441 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     7501 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     7561 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     7621 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     7681 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     7741 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     7801 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     7861 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     7921 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     7981 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     8041 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     8101 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     8161 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     8221 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     8281 caaaggaata agccaaggaa cctctggatt acaaaatttg tgaaagattg actggtattc
     8341 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcagt
     8401 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     8461 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     8521 ttatcatgtc tggaattgac tcaaatgatg tcaattagtc tatcagaagc tcatctggtc
     8581 tcccttccgg gggacaagac atccctgttt aatatttaaa cagcagtgtt cccaaactgg
     8641 gttcttatat cccttgctct ggtcaaccag gttgcagggt ttcctgtcct cacaggaacg
     8701 aagtccctaa agaaacagtg gcagccaggt ttagccccgg aattgactgg attccttttt
     8761 tagggcccat tggtatggct ttttccccgt atccccccag gtgtctgcag gctcaaagag
     8821 cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc
     8881 ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg
     8941 gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg
     9001 gggctgtccc tgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag
     9061 aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata
     9121 atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca
     9181 ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg
     9241 acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta
     9301 cgcagactat ctttctaggg ttaatctagc tgcatcagga tcatatcgtc gggtcttttt
     9361 tccggctcag tcatcgccca agctggcgct atctgggcat cggggaggaa gaagcccgtg
     9421 ccttttcccg cgaggttgaa gcggcatgga aagagtttgc cgaggatgac tgctgctgca
     9481 ttgacgttga gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg gccatgcacg
     9541 cctttaacgg tgaactgttc gttcaggcca cctgggatac cagttcgtcg cggcttttcc
     9601 ggacacagtt ccggatggtc agcccgaagc gcatcagcaa cccgaacaat accggcgaca
     9661 gccggaactg ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg ggatattacg
     9721 tcagcgagga cgggtatcct ggctggatgc cgcagaaatg gacatggata ccccgtgagt
     9781 tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     9841 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     9901 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     9961 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
    10021 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
    10081 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
    10141 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
    10201 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
    10261 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
    10321 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
    10381 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
    10441 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
    10501 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
    10561 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
    10621 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
    10681 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
    10741 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
    10801 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
    10861 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
    10921 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
    10981 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
    11041 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
    11101 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
    11161 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
    11221 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
    11281 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
    11341 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
    11401 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
    11461 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
    11521 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
    11581 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
    11641 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
    11701 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
    11761 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
    11821 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
    11881 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
    11941 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
    12001 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
    12061 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
    12121 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
    12181 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
    12241 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
    12301 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
    12361 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
    12421 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
    12481 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
    12541 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcctcgtt cattcacgtt
    12601 tttgaacccg tggaggacgg gcagactcgc ggtgcaaatg tgttttacag cgtgatggag
    12661 cagatgaaga tgctcgacac gctgcagaac acgcagctag attaacccta gaaagataat
    12721 catattgtga cgtacgttaa agataatcat gtgtaaaatt gacgcatgtg ttttatcggt
    12781 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
    12841 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
    12901 acaaaaactc aaaatttctt ctataaagta acaaaacttt tatgagggac agcccccccc
    12961 caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc
    13021 ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca
    13081 gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt
    13141 tgagcctgca gacacctggg gggatacggg gaaaaggcct ccacggccac tagtctttcg
    13201 tcttcaagaa ttcctcgagt ttactcccta tcagtgatag agaacgtatg aagagtttac
    13261 tccctatcag tgatagagaa cgtatgcaga ctttactccc tatcagtgat agagaacgta
    13321 taaggagttt actccctatc agtgatagag aacgtatgac cagtttactc cctatcagtg
    13381 atagagaacg tatctacagt ttactcccta tcagtgatag agaa
//
